Stabilized soluble pre-fusion RSV F proteins

ABSTRACT

Stable pre-fusion respiratory syncitial virus (RSV) F proteins (or fragment thereof) are described. Compositions containing the proteins and uses of the compositions for the prevention and/or treatment of RSV infection are also described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Application No.PCT/EP2017/057962, filed Apr. 4, 2017, which was published in theEnglish language on Oct. 12, 2017, under International Publication No.WO 2017/174568 A1 which claims priority under 35 U.S.C. § 119(b) toEuropean Application No. 16163810.1, filed Apr. 5, 2016, the disclosuresof which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence Listing 688097 535US”, creation date of Oct. 3,2018, and having a size of about 31.8 KB. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

The present invention relates to the field of medicine. The invention inparticular relates to recombinant pre-fusion RSV F proteins and usesthereof; e.g. as a vaccine.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a highly contagious childhoodpathogen of the respiratory tract which is believed to be responsiblefor 200,000 childhood deaths annually. In children younger than 2 years,RSV accounts for approximately 50% of the hospitalizations due torespiratory infections, with a peak of hospitalization occurring at 2-4months of age. It has been reported that almost all children will haveexperienced infection with RSV by the age of two, and repeated infectionduring life is attributed to low natural immunity. In the elderly, theRSV disease burden is similar to those caused by non-pandemic influenzaA infections.

To infect a host cell, RSV, like other enveloped viruses such asinfluenza virus and HIV, require fusion of the viral membrane with ahost cell membrane. For RSV the conserved fusion protein (RSV F protein)fuses the viral and host cell cellular membranes. In current models,based on paramyxovirus studies, the RSV F protein initially folds into a“pre-fusion” conformation. The metastable structure has recently beensolved in complex with a stabilizing neutralizing antibody Fab fragment(McLellan et al., Science 340(6136):1113-7, 2013). During cell entry,the pre-fusion conformation undergoes refolding and conformationalchanges to its “post-fusion” conformation (McLellan, J. Virol85(15):7788-96, 2010; Swanson, PNAS 108(23):9619-24, 2011). Thus, theRSV F protein is a metastable protein that drives membrane fusion bycoupling irreversible protein refolding to membrane juxtaposition byinitially folding into a metastable form (pre-fusion conformation) thatsubsequently undergoes discrete/stepwise conformational changes to alower energy conformation (post-fusion conformation). These observationssuggest that pre-fusion and post-fusion RSV F protein are antigenicallydistinct (Calder, L. J. et al. Virology 271, 122-131 (2000)). It isclear from electron microscopy of RSV-F that large structuraldifferences between the pre-fusion and post-fusion F trimer exist, whichhas recently been confirmed by crystallography (McLellan J. S. et al.Science 340(6136):1113-7 (2013) and McLellan J. S. et al. Science342(6158): 592-8 (2013)) and it was shown that most of the neutralizingantibodies in the serum of RSV-positive individuals are binding topre-fusion F (Ngwuta et. al., Science Translational Medicine, 7(309):309ra162, 1-9).

A vaccine against RSV infection is not currently available, but isdesired. Vaccine candidates based on the RSV F protein have failed dueto problems with e.g. stability, purity, reproducibility, and potency.As indicated above, crystal structures have revealed a largeconformational change between the pre-fusion and post-fusion states. Themagnitude of the rearrangement suggested that only a portion ofantibodies directed to the post-fusion conformation of RSV-F will beable to cross react with the native conformation of the pre-fusion spikeon the surface of the virus. Accordingly, efforts to produce a vaccineagainst RSV have focused on developing vaccines that contain pre-fusionforms of RSV F protein (see, e.g., WO20101149745, WO2010/1149743,WO2009/1079796, WO2012/158613). However, these efforts have not yetyielded stable pre-fusion RSV F proteins that could be used ascandidates for testing in humans.

Therefore, a need remains for efficient vaccines and methods ofvaccinating against RSV, in particular comprising RSV F proteins in thepre-fusion conformation. The present invention aims at providing suchvaccines and methods for vaccinating against RSV in a safe andefficacious manner.

SUMMARY OF THE INVENTION

The present invention provides stable, recombinant, pre-fusionrespiratory syncytial virus (RSV) fusion (F) proteins, i.e. recombinantRSV F proteins in soluble form (i.e. not membrane bound) that arestabilized in the pre-fusion conformation, wherein the RSV F proteincomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or fragments thereof.

In certain embodiments, the RSV F proteins, or fragments thereof,comprise at least one epitope that is specific to the pre-fusionconformation F protein, wherein the at least one epitope is recognizedby a pre-fusion specific monoclonal antibody comprising a heavy chainCDR1 region of SEQ ID NO: 4, a heavy chain CDR2 region of SEQ ID NO: 5,a heavy chain CDR3 region of SEQ ID NO: 6 and a light chain CDR1 regionof SEQ ID NO: 7, a light chain CDR2 region of SEQ ID NO: 8, and a lightchain CDR3 region of SEQ ID NO: 9, and/or a pre-fusion specificmonoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO:10, a heavy chain CDR2 region of SEQ ID NO: 11, a heavy chain CDR3region of SEQ ID NO: 12 and a light chain CDR1 region of SEQ ID NO: 13,a light chain CDR2 region of SEQ ID NO: 14, and a light chain CDR3region of SEQ ID NO: 15.

In certain embodiments, the RSV F proteins are trimeric.

The invention also provides nucleic acid molecules encoding thepre-fusion RSV F proteins or fragments thereof according to theinvention and vectors comprising such nucleic acid molecules.

The invention also relates to compositions, preferably immunogeniccompositions, comprising said RSV pre-fusion F protein (or fragmentsthereof), nucleic acid molecule encoding said RSV pre-fusion F protein,and to the use thereof in inducing an immune response against RSV Fprotein, in particular to the use thereof as a vaccine. The inventionalso relates to methods for inducing an anti-respiratory syncytial virus(RSV) immune response in a subject, comprising administering to thesubject an effective amount of a pre-fusion RSV F protein, a nucleicacid molecule encoding said RSV F protein, and/or a vector comprisingsaid nucleic acid molecule. Preferably, the induced immune response ischaracterized by neutralizing antibodies to RSV and/or protectiveimmunity against RSV. In particular aspects, the invention relates to amethod for inducing neutralizing anti-respiratory syncytial virus (RSV)F protein antibodies in a subject, comprising administering to thesubject an effective amount of an immunogenic composition comprising apre-fusion RSV F protein, a nucleic acid molecule encoding said RSV Fprotein, and/or a vector comprising said nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of RSV F variants. SCDM—single-chaindouble mutant, SCTM—single-chain triple mutant, PRQM—processed quadruplemutant and PRPM—processed penta-mutant. Secreted proteins are presentedwithout signal peptide and p27 fragment. F1 and F2 domains areindicated, as well as fusion peptide (FP), fibritin trimerization domain(foldon) and the linker in single-chain proteins between F2 and F1(GSGSG). Three stabilizing mutations (N67I, S215P and D386N) (blackdiamonds). Two mutations to improve antigenic match to circulatingstrains (K66E and I76V) (grey diamonds). The residue position isnumbered as in the full length wild type protein including signalpeptide.

FIG. 2. Protein expression levels and pre-fusion stability of processedRSV F PR-A2 variants with multiple amino acid substitutions. Proteinexpression levels in cell culture supernatants were tested 72 hours posttransfection by quantitative octet (Q-Octet) with CR9501 and CR9503(bars to the left) and fraction of RSV F protein binding to pre-fusionspecific CR9501 antibody on the day of harvest and after storage at 4°C. for indicated period of time (bars to the right). Bars representaverage of 2-4 measurements, lines represent range of values.

FIG. 3. Melting temperatures (Tm) of the purified RSV-F proteins. Eachmeasurement is represented by a dot.

FIG. 4. K66E and I76V amino acid substitutions did not have effect on Fprotein expression levels and pre-fusion stability. Protein expressionlevels in cell culture supernatants were tested 96 hours posttransfection by Q-Octet with CR9501 and CR9503 (bars to the left) andfraction of RSV F protein binding to pre-fusion specific CR9501 antibodyon the day of harvest and after storage at 4° C. for indicated period oftime (bars to the right). Bars represent average of 2 measurements,lines represent range of values.

FIG. 5: Pre-fusion stability of the F protein variants in CHO cellculture supernatant. Protein expression levels in cell culturesupernatants were tested 96 hours post transfection by Q-Octet withCR9501 and CR9503 and fraction of RSV F protein binding to pre-fusionspecific CR9501 antibody on the day of harvest and after storage at 4°C. for indicated period of time. Bars represent average of 2measurements, lines represent range of values. PRQM—PR-A2 with N67I,S215P, K66E, and I76V; PRPM—PR-A2 with N67I, S215P, K66E, I76V andD486N.

FIG. 6: RSV F proteins of the invention stay intact in CHO cell culturesupernatant at pH5. pH of the cell culture supernatants containing Fprotein variants was adjusted to pH5 and the samples were incubated at 7days with or without protease inhibitors. The samples were analyzed onSDS-PAGE under reducing conditions. The first lane of each gel ismolecular weight standard marker; the size of the standard proteins isindicated. The samples: 1—day 0 sample; 2—day 7 sample incubated at 4°C.; 3—day 7 sample incubated at 4° C. with protease inhibitors; 4—day 0sample; 5—day 7 sample incubated at room temperature; 6—day 7 sampleincubated at room temperature with protease inhibitors; 7—day 0 sample;8—day 7 sample incubated at 37° C.; 9—day 7 sample incubated at 37° C.with protease inhibitors. In the processed protein samples, the lowerband represents the F1 domain and the upper band represents partiallyprocessed protein (F1+p27) or unprocessed protein F1+F2). In thesingle-chain protein sample, the band is F1+F2 domains. PRQM—PR-A2 withN67I, S215P, K66E, and I76V; PRPM—PR-A2 with N67I, S215P, K66E, I76V andD486N. LNR: K683-065.

FIG. 7 Temperature stability of RSV F proteins in CHO cell culturesupernatant. The supernatant samples were subjected to heat treatmentfor 30 min at temperatures 45-65° C. The amount of pre-fusion protein inthe sample was measured in ELISA with CR9501 antibodies. The values werenormalized to untreated sample (20° C.). The curves are shown for eachprotein individually and an overlay of all curves (on the lower right).Each point represents a replicate measurement. Two assays were performedwith 2 technical replicates each. The curves were fitted using Nonlinearregression variable slope equation (GraphPad Prism); meltingtemperatures (Tm) were calculated as IC50 values. PRQM—PR-A2 with N67I,S215P, K66E, and I76V; PRPM—PR-A2 with N67I, S215P, K66E, I76V andD486N.

FIG. 8: RSV titers in lungs and nose 5 days after challenge with RSV A2.RSV titers in lungs (upper panel) and nose (lower panel) 5 days afterchallenge with RSV A2. The lower level of detection (LOD) is indicatedby a dotted line. Mean titers (log 10 pfu per gram of tissue) areindicated with horizontal bars. Adjuvanted and non-adjuvanted PRPMgroups were compared across dose by a Cochran-Mantel-Haenszel test andstatistical differences are indicated in the figure. i.m.:intramuscular; i.n: intranasal.

FIG. 9: RSV neutralizing titers against RSV A Long in cotton rats seraat day 49 after priming. RSV neutralizing titers (IC50 (log 2)) againstRSV A Long using an ELISA-based readout were determined in cotton ratssera at day 49 after priming. The mean of each group is indicated with ahorizontal bar. The limit of detection (LOD) is set on 3.0 (log 2 andindicated with a dashed line). VNA titers induced PRPM by adjuvanted andnon-adjuvanted were compared across dose by ANOVA and the results areindicated in the figure. i.m.: intramuscular; i.n: intranasal.

DETAILED DESCRIPTION OF THE INVENTION

The fusion protein (F) of the respiratory syncictial virus (RSV) isinvolved in fusion of the viral membrane with a host cell membrane,which is required for infection. The RSV F mRNA is translated into a 574amino acid precursor protein designated F0, which contains a signalpeptide sequence of 26 amino acids at the N-terminus that is removed bya signal peptidase in the endoplasmic reticulum. F0 is cleaved at twosites (between amino acid residues 109/110 and 136/137) by cellularfurin-like proteases in the trans-Golgi, removing a short glycosylatedintervening sequence (also referred to a p27 region, comprising theamino acid residues 110 to 136, and generating two domains or subunitsdesignated F1 and F2. The F1 domain (amino acid residues 137-574)contains a hydrophobic fusion peptide at its N-terminus and theC-terminus contains the transmembrane (TM) (amino acid residues 530-550)and cytoplasmic region (amino acid residues 551-574). The F2 domain(amino acid residues 27-109) is covalently linked to F1 by two disulfidebridges. The F1-F2 heterodimers are assembled as homotrimers in thevirion.

A vaccine against RSV infection is not currently available, but isdesired. One potential approach to producing a vaccine is a subunitvaccine based on purified RSV F protein. However, for this approach itis desirable that the purified RSV F protein is in a conformation whichresembles the conformation of the pre-fusion state of RSV F protein, andwhich is stable over time, and can be produced in sufficient quantities.In addition, for a subunit-based vaccine, the RSV F protein needs to betruncated by deletion of the transmembrane (TM) and the cytoplasmicregion to create a soluble secreted F protein (sF). Because the TMregion is responsible for membrane anchoring and trimerization, theanchorless soluble F protein is considerably more labile than thefull-length protein and will readily refold into the post-fusionend-state. In order to obtain soluble F protein in the stable pre-fusionconformation that shows high expression levels and high stability, thepre-fusion conformation thus needs to be stabilized.

Several mutations stabilizing RSV F protein in the pre-fusionconformation have previously been described in WO2014/174018 andWO2014/202570. The RSV F proteins according to the present inventioncomprise a unique and specific subset of mutations described earlier incombination with two further mutations. According to the invention ithas been shown that this unique combination of mutations of the presentinvention results in increased RSV F protein expression levels andstability of the pre-fusion conformation.

The present invention thus provides novel stable soluble pre-fusion RSVF proteins, i.e. soluble RSV F proteins that are stabilized in thepre-fusion conformation, or fragments thereof. The RSV F proteinsaccording to the present invention comprise an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQID NO: 3.

In the research that led to the present invention, a unique combinationof mutations was introduced together with a heterologous trimerizationdomain in order to obtain said stable soluble pre-fusion RSV F proteins.The stable pre-fusion RSV F proteins of the invention are in thepre-fusion conformation, i.e. they comprise (display) at least oneepitope that is specific to the pre-fusion conformation F protein. Anepitope that is specific to the pre-fusion conformation F protein is anepitope that is not presented in the post-fusion conformation. Withoutwishing to be bound by any particular theory, it is believed that thepre-fusion conformation of RSV F protein may contain epitopes that arethe same as those on the RSV F protein expressed on natural RSV virions,and therefore may provide advantages for eliciting protectiveneutralizing antibodies.

In certain embodiments, the RSV pre-fusion F proteins (or fragmentsthereof) of the invention comprise at least one epitope that isrecognized by a pre-fusion specific monoclonal antibody, comprising aheavy chain CDR1 region of SEQ ID NO: 4, a heavy chain CDR2 region ofSEQ ID NO: 5, a heavy chain CDR3 region of SEQ ID NO: 6 and a lightchain CDR1 region of SEQ ID NO: 7, a light chain CDR2 region of SEQ IDNO: 8, and a light chain CDR3 region of SEQ ID NO: 9 (hereafter referredto as CR9501) and/or a pre-fusion specific monoclonal antibody,comprising a heavy chain CDR I region of SEQ ID NO: 10, a heavy chainCDR2 region of SEQ ID NO: 11, a heavy chain CDR3 region of SEQ ID NO: 12and a light chain CDR1 region of SEQ ID NO: 13, a light chain CDR2region of SEQ ID NO: 14, and a light chain CDR3 region of SEQ ID NO: 15(referred to as CR9502). CR9501 and CR9502 comprise the heavy and lightchain variable regions, and thus the binding specificities, of theantibodies 58C5 and 30D8, respectively, which have previously been shownto bind specifically to RSV F protein in its pre-fusion conformation andnot to the post-fusion conformation (as disclosed in WO2012/006596).

In certain embodiments, the recombinant pre-fusion RSV F proteins aretrimeric.

As used throughout the present application nucleotide sequences areprovided from 5′ to 3′ direction, and amino acid sequences fromN-terminus to C-terminus, as custom in the art.

As indicated above, fragments of the pre-fusion RSV F protein are alsoencompassed by the present invention. The fragment may result fromeither or both of amino-terminal (e.g. by cleaving off the signalsequence) and carboxy-terminal deletions. The fragment may be chosen tocomprise an immunologically active fragment of the F protein, i.e. apart that will give rise to an immune response in a subject. This can beeasily determined using in silico, in vitro and/or in vivo methods, allroutine to the skilled person.

In certain embodiments, the encoded proteins according to the inventioncomprise a signal sequence, also referred to as leader sequence orsignal peptide, corresponding to amino acids 1-26 of SEQ ID NO: 1, SEQID NO: 2 or SEQ ID NO: 3. Signal sequences typically are short (e.g.5-30 amino acids long) amino acid sequences present at the N-terminus ofthe majority of newly synthesized proteins that are destined towards thesecretory pathway, and are typically cleaved by signal peptidase togenerate a free signal peptide and a mature protein.

In certain embodiments, the proteins according to the invention do notcomprise a signal sequence.

The present invention further provides nucleic acid molecules encodingthe RSV pre-fusion F proteins, or fragments thereof, according to theinvention.

In preferred embodiments, the nucleic acid molecules encoding the RSV Fproteins according to the invention are codon-optimized for expressionin mammalian cells, preferably human cells. Methods ofcodon-optimization are known and have been described previously (e.g. WO96/09378). A sequence is considered codon-optimized if at least onenon-preferred codon as compared to a wild type sequence is replaced by acodon that is more preferred. Herein, a non-preferred codon is a codonthat is used less frequently in an organism than another codon codingfor the same amino acid, and a codon that is more preferred is a codonthat is used more frequently in an organism than a non-preferred codon.The frequency of codon usage for a specific organism can be found incodon frequency tables, such as in http://www.kazusa.or.jp/codon.Preferably more than one non-preferred codon, preferably most or allnon-preferred codons, are replaced by codons that are more preferred.Preferably the most frequently used codons in an organism are used in acodon-optimized sequence. Replacement by preferred codons generallyleads to higher expression.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acid molecules can encode the same proteinas a result of the degeneracy of the genetic code. It is also understoodthat skilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the protein sequence encoded by thenucleic acid molecules to reflect the codon usage of any particular hostorganism in which the proteins are to be expressed. Therefore, unlessotherwise specified, a “nucleotide sequence encoding an amino acidsequence” includes all nucleotide sequences that are degenerate versionsof each other and that encode the same amino acid sequence. Nucleotidesequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biologytechniques, or generated de novo by DNA synthesis, which can beperformed using routine procedures by service companies having businessin the field of DNA synthesis and/or molecular cloning (e.g. GeneArt,GenScripts, Invitrogen, Eurofins).

In certain embodiments, the nucleic acid molecules comprise a nucleotidesequence of SEQ ID NO. 2221, or 22.

The invention also provides vectors comprising a nucleic acid moleculeas described above. In certain embodiments, a nucleic acid moleculeaccording to the invention thus is part of a vector. Such vectors caneasily be manipulated by methods well known to the person skilled in theart, and can for instance be designed for being capable of replicationin prokaryotic and/or eukaryotic cells. In addition, many vectors can beused for transformation of eukaryotic cells and will integrate in wholeor in part into the genome of such cells, resulting in stable host cellscomprising the desired nucleic acid in their genome. The vector used canbe any vector that is suitable for cloning DNA and that can be used fortranscription of a nucleic acid of interest. The person skilled in theart is capable of choosing suitable expression vectors, and insertingthe nucleic acid sequences of the invention in a functional manner.

Host cells comprising the nucleic acid molecules encoding the pre-fusionRSV F proteins form also part of the invention. The pre-fusion RSV Fproteins may be produced through recombinant DNA technology involvingexpression of the molecules in host cells, e.g. Chinese hamster ovary(CHO) cells, tumor cell lines, BHK cells, human cell lines such asHEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like,or transgenic animals or plants. In certain embodiments, the cells arefrom a multicellular organism, in certain embodiments they are ofvertebrate or invertebrate origin. In certain embodiments, the cells aremammalian cells. In certain embodiments, the cells are human cells. Ingeneral, the production of a recombinant proteins, such the pre-fusionRSV F proteins of the invention, in a host cell comprises theintroduction of a heterologous nucleic acid molecule encoding theprotein in expressible format into the host cell, culturing the cellsunder conditions conducive to expression of the nucleic acid moleculeand allowing expression of the protein in said cell. The nucleic acidmolecule encoding a protein in expressible format may be in the form ofan expression cassette, and usually requires sequences capable ofbringing about expression of the nucleic acid, such as enhancer(s),promoter, polyadenylation signal, and the like. The person skilled inthe art is aware that various promoters can be used to obtain expressionof a gene in host cells. Promoters can be constitutive or regulated, andcan be obtained from various sources, including viruses, prokaryotic, oreukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitablemedium can be routinely chosen for a host cell to express the protein ofinterest, here the pre-fusion RSV F proteins. The suitable medium may ormay not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as‘transgene’) is a nucleic acid molecule that is not naturally present inthe host cell. It is introduced into for instance a vector by standardmolecular biology techniques. A transgene is generally operably linkedto expression control sequences. This can for instance be done byplacing the nucleic acid encoding the transgene(s) under the control ofa promoter. Further regulatory sequences may be added. Many promoterscan be used for expression of a transgene(s), and are known to theskilled person, e.g. these may comprise viral, mammalian, syntheticpromoters, and the like. A non-limiting example of a suitable promoterfor obtaining expression in eukaryotic cells is a CMV-promoter (U.S.Pat. No. 5,385,839), e.g. the CMV immediate early promoter, for instancecomprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter. A polyadenylation signal, for example the bovinegrowth hormone polyA signal (U.S. Pat. No. 5,122,458), may be presentbehind the transgene(s). Alternatively, several widely used expressionvectors are available in the art and from commercial sources, e.g. thepcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BDSciences, pCMV-Script from Stratagene, etc, which can be used torecombinantly express the protein of interest, or to obtain suitablepromoters and/or transcription terminator sequences, polyA sequences,and the like.

The cell culture can be any type of cell culture, including adherentcell culture, e.g. cells attached to the surface of a culture vessel orto microcarriers, as well as suspension culture. Most large-scalesuspension cultures are operated as batch or fed-batch processes becausethey are the most straightforward to operate and scale up. Nowadays,continuous processes based on perfusion principles are becoming morecommon and are also suitable. Suitable culture media are also well knownto the skilled person and can generally be obtained from commercialsources in large quantities, or custom-made according to standardprotocols. Culturing can be done for instance in dishes, roller bottlesor in bioreactors, using batch, fed-batch, continuous systems and thelike. Suitable conditions for culturing cells are known (see e.g. TissueCulture, Academic Press, Kruse and Paterson, editors (1973), and R. I.Freshney, Culture of animal cells: A manual of basic technique, fourthedition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion RSVF protein and/or a nucleic acid molecule, and/or a vector, as describedabove. The invention thus provides compositions comprising a pre-fusionRSV F protein that displays an epitope that is present in a pre-fusionconformation of the RSV F protein but is absent in the post-fusionconformation, or a fragment thereof. The invention also providescompositions comprising a nucleic acid molecule and/or a vector,encoding such pre-fusion RSV F protein or fragment thereof. Thecompositions preferably are immunogenic compositions comprising apre-fusion RSV F protein, and/or a nucleic acid molecule, and/or avector, as described above. The invention also provides the use of astabilized pre-fusion RSV F protein or a nucleic acid molecule encodingsaid RSV F protein according to the invention, for inducing an immuneresponse against RSV F protein in a subject. Further provided aremethods for inducing an immune response against RSV F protein in asubject, comprising administering to the subject a pre-fusion RSV Fprotein, and/or a nucleic acid molecule, and/or a vector, according tothe invention. Also provided are pre-fusion RSV F proteins, nucleic acidmolecules, and/or vectors, according to the invention for use ininducing an immune response against RSV F protein in a subject. Furtherprovided is the use of the pre-fusion RSV F proteins, and/or nucleicacid molecules, and/or vectors according to the invention for themanufacture of a medicament for use in inducing an immune responseagainst RSV F protein in a subject.

The pre-fusion RSV F proteins, nucleic acid molecules, or vectors of theinvention may be used for prevention (prophylaxis) and/or treatment ofRSV infections. In certain embodiments, the prevention and/or treatmentmay be targeted at patient groups that are susceptible RSV infection.Such patient groups include, but are not limited to e.g., the elderly(e.g. ≥50 years old, ≥60 years old, and preferably ≥65 years old), theyoung (e.g. ≤5 years old, ≤1 year old), hospitalized patients andpatients who have been treated with an antiviral compound but have shownan inadequate antiviral response.

The pre-fusion RSV F proteins, nucleic acid molecules and/or vectorsaccording to the invention may be used e.g. in stand-alone treatmentand/or prophylaxis of a disease or condition caused by RSV, or incombination with other prophylactic and/or therapeutic treatments, suchas (existing or future) vaccines, antiviral agents and/or monoclonalantibodies.

The invention further provides methods for preventing and/or treatingRSV infection in a subject utilizing the pre-fusion RSV F proteins,nucleic acid molecules and/or vectors according to the invention. In aspecific embodiment, a method for preventing and/or treating RSVinfection in a subject comprises administering to a subject in needthereof an effective amount of a pre-fusion RSV F protein, nucleic acidmolecule and/or a vector, as described above. A therapeuticallyeffective amount refers to an amount of a protein, nucleic acid moleculeor vector, which is effective for preventing, ameliorating and/ortreating a disease or condition resulting from infection by RSV.Prevention encompasses inhibiting or reducing the spread of RSV orinhibiting or reducing the onset, development or progression of one ormore of the symptoms associated with infection by RSV. Amelioration asused in herein may refer to the reduction of visible or perceptibledisease symptoms, viremia, or any other measurable manifestation ofinfluenza infection.

For administering to subjects, such as humans, the invention may employpharmaceutical compositions comprising a pre-fusion RSV F protein, anucleic acid molecule and/or a vector as described herein, and apharmaceutically acceptable carrier or excipient. In the presentcontext, the term “pharmaceutically acceptable” means that the carrieror excipient, at the dosages and concentrations employed, will not causeany unwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The RSV F proteins, or nucleic acid molecules, preferablyare formulated and administered as a sterile solution although it mayalso be possible to utilize lyophilized preparations. Sterile solutionsare prepared by sterile filtration or by other methods known per se inthe art. The solutions are then lyophilized or filled intopharmaceutical dosage containers. The pH of the solution generally is inthe range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The RSV F proteinstypically are in a solution having a suitable pharmaceuticallyacceptable buffer, and the composition may also contain a salt.Optionally stabilizing agent may be present, such as albumin. In certainembodiments, detergent is added. In certain embodiments, the RSV Fproteins may be formulated into an injectable preparation.

In certain embodiments, a composition according to the invention furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the RSV F proteins of theinvention. Examples of suitable adjuvants include aluminium salts suchas aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, and the like; eukaryotic proteins (e.g. antibodies orfragments thereof (e.g. directed against the antigen itself or CD1a,CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc),which stimulate immune response upon interaction with recipient cells.In certain embodiments the compositions of the invention comprisealuminium as an adjuvant, e.g. in the form of aluminium hydroxide,aluminium phosphate, aluminium potassium phosphate, or combinationsthereof, in concentrations of 0.05-5 mg, e.g. from 0.075-1.0 mg, ofaluminium content per dose.

The pre-fusion RSV F proteins may also be administered in combinationwith or conjugated to nanoparticles, such as e.g. polymers, liposomes,virosomes, virus-like particles or self-assembling protein particles.The pre-fusion F proteins may be combined with, encapsidated in orconjugated to the nanoparticles with or without adjuvant. Encapsulationwithin liposomes is described, e.g. in U.S. Pat. No. 4,235,877.Conjugation to macromolecules is disclosed, for example in U.S. Pat. No.4,372,945 or 4,474,757.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the invention provides methods for making avaccine against respiratory syncytial virus (RSV), comprising providinga composition according to the invention and formulating it into apharmaceutically acceptable composition. The term “vaccine” refers to anagent or composition containing an active component effective to inducea certain degree of immunity in a subject against a certain pathogen ordisease, which will result in at least a decrease (up to completeabsence) of the severity, duration or other manifestation of symptomsassociated with infection by the pathogen or the disease. In the presentinvention, the vaccine comprises an effective amount of a pre-fusion RSVF protein and/or a nucleic acid molecule encoding a pre-fusion RSV Fprotein, and/or a vector comprising said nucleic acid molecule, whichresults in an immune response against the F protein of RSV. Thisprovides a method of preventing serious lower respiratory tract diseaseleading to hospitalization and the decrease in frequency ofcomplications such as pneumonia and bronchiolitis due to RSV infectionand replication in a subject. The term “vaccine” according to theinvention implies that it is a pharmaceutical composition, and thustypically includes a pharmaceutically acceptable diluent, carrier orexcipient. It may or may not comprise further active ingredients. Incertain embodiments it may be a combination vaccine that furthercomprises other components that induce an immune response, e.g. againstother proteins of RSV and/or against other infectious agents. Theadministration of further active components may for instance be done byseparate administration or by administering combination products of thevaccines of the invention and the further active components.

Compositions may be administered to a subject, e.g. a human subject. Thetotal dose of the RSV F proteins in a composition for a singleadministration can for instance be about 0.01 μg to about 10 mg, e.g. 1μg-1 mg, e.g. 10 μg-100 μg. Determining the recommended dose will becarried out by experimentation and is routine for those skilled in theart.

Administration of the compositions according to the invention can beperformed using standard routes of administration. Non-limitingembodiments include parenteral administration, such as intradermal,intramuscular, subcutaneous, transcutaneous, or mucosal administration,e.g. intranasal, oral, and the like. In one embodiment a composition isadministered by intramuscular injection. The skilled person knows thevarious possibilities to administer a composition, e.g. a vaccine inorder to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent,e.g. a mouse, a cotton rat, or a non-human-primate, or a human.Preferably, the subject is a human subject.

The proteins, nucleic acid molecules, vectors, and/or compositions mayalso be administered, either as prime, or as boost, in a homologous orheterologous prime-boost regimen. If a boosting vaccination isperformed, typically, such a boosting vaccination will be administeredto the same subject at a time between one week and one year, preferablybetween two weeks and four months, after administering the compositionto the subject for the first time (which is in such cases referred to as‘priming vaccination’). In certain embodiments, the administrationcomprises a prime and at least one booster administration.

In addition, the proteins of the invention may be used as diagnostictool, for example to test the immune status of an individual byestablishing whether there are antibodies in the serum of suchindividual capable of binding to the protein of the invention. Theinvention thus also relates to an in vitro diagnostic method fordetecting the presence of an RSV infection in a patient said methodcomprising the steps of a) contacting a biological sample obtained fromsaid patient with a protein according to the invention; and b) detectingthe presence of antibody-protein complexes.

EXAMPLES Example 1: Generation of the Stable Pre Fusion RSV F Protein

Several pre-fusion RSV F protein variants were produced, which areschematically represented in FIG. 1. All candidates comprise a fibritintrimerization domain (foldon) (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO:20), linked to the amino acid residue 495 of the RSV A2 F1 domain.

In the processed versions of RSV F (i.e. the versions which are cleavedremoving the p27 region) the N67I substitution had the strongest effecton both the expression level and stability but fully stable pre-fusion Fprotein was obtained only when the 67 and 215 substitutions werecombined, resulting in a 20-fold expression level increase (FIG. 2).Addition of a third amino acid substitution did not improve expressionlevel or stability as measured by storage stability at 4° C. However,when the RSV F proteins were purified and further characterized, itturned out that the extra third substitution significantly stabilizesthe pre-fusion F protein as measured by the more stringent temperaturestability test (by Differential Scanning Fluorimetry assay—DSF) (FIG.3).

Because the A2 strain that was used as a parental sequence for the RSV Fprotein variants described previously (WO2014/174018 and WO2014/202570)is a cell line adapted laboratory strain which had accumulated twounique and rare mutations in the apex K66 and 176), it was decided tomutate these two residues to match the natural clinical isolates (K66E,I76V). The K66E and I76V mutations were included in the new processedprotein design to make the sequence closer to the natural virusisolates. The K66E+I76V substitutions were tested in selected stabilizedvariants to demonstrate that the amino acid substitutions did not havenegative effect on protein expression or stability. It was shown thatthe proteins were stable in cell culture supernatants for longer than 2weeks. There was no negative effect on the expression level of the Fproteins, on the contrary, RSV F protein with N67I, S215P, K66E and 176Vmutations expressed to a higher level than protein with only N67I andS215P (FIG. 4).

The processed RSV F proteins with N67I, S215P, K66E and I76V (named PRQMfor processed quadruple-mutant) and with N67I, S215P, K66E, I76V andD486N (named PRPM for processed penta-mutant) were purified and furthercharacterized.

The screening of the stabilizing mutations for the RSV F protein wasperformed in suspension HEK cells (FreeStyle 293F). These cells areconvenient to use in a research laboratory because they are adapted tosimple transfection protocol and express proteins at a high level. Forbig scale and GMP protein production CHO cells are often the cell lineof choice. Therefore expression and stability of several preferred Fprotein designs was tested in suspension CHO cells (FreeStyle CHO-S).CHO-S cells are difficult to transfect and therefore overall expressionlevels were expected to be lower than in HEK cells. During analysistherefore we focused on relative expression of the proteins and theirstability.

Five processed proteins were selected for the test. The 5 variants allcontained the substitutions K66E, I76V, N67I and S215P. As describedabove, the latter 2 are required to stabilize the protein in pre-fusionconformation; the former two were included to make the sequence closerto naturally occurring isolates (as was described in the previoussection). The proteins differed by the additional mutations E161P, D486Nand E487Q. These were chosen because of high expression level, storagestability and low impact on antigenicity. All proteins were expressed inCHO cells and had comparable storage stability. The RSV F proteins werestable in pre-fusion conformation when stored in cell culturesupernatants for 2 weeks at 4° C. (FIG. 5). Also, the stability of theRSV F proteins in CHO cell culture supernatant at pH5 was tested. Asshown in FIG. 6 no degradation after incubation of protein samples for 7days at different temperatures was detected.

In conclusion, the RSV F proteins of the invention expressed in CHOcells and were stable in cell culture supernatants. Additionally, thetemperature stability of the protein was tested. The cell culturesupernatants were subjected to heat treatment and amount of pre-fusionprotein in the samples was measured in ELISA with CR9501 antibody (FIG.7).

The variant with D486N (PRPM protein) was most stable againsttemperature stress. Addition of K498R mutation seemed to have noadvantage compared to protein with minimal amount of modification(PRQM). The variants with E161P mutation had highest expression levels(data not shown). However the drawback of this amino acid substitutionwas that the residue 161 is located on the surface of the protein and onthe fringe of epitope for CR9501 antibody.

According to the present invention, it thus was shown that the PRPM (RSVF protein with fibritin foldon trimerization domain and with mutationsN67I, S215P, K66E, I76V and D486N, SEQ ID NO: 1) and the PRQM (RSV Fprotein with fibritin foldon trimerization domain and with N67I, S215P,K66E, and I76V, SEQ ID NO: 2) as a processed pre-fusion protein withminimum of required sequence modifications, as well as the PRQM +S46G orPRPM +S46G variant all are stabilized in the pre-fusion conformation andshow a high Tm (Table 1). The latter variants with the S46G substitutionhave a significantly higher expression level.

TABLE 1 Protein ID Freeze-thaw stability Tm (° C.) PRQM S46G Stable for3 cycles, 56.2 aggregation after 5 cycles PRPM S46G Stable for 5 cyles63.6 PRPM Stable for 5 cycles 65.0

Example 2: Immunogenicity and Protection Induced by PRPM with andwithout Adjuvant

An experiment was conducted to determine the immunogenic andprophylactic efficacy of the recombinant PRPM protein in the presence orabsence of an adjuvant in a homologous RSV-A2 challenge cotton ratmodel. The animals were immunized i.m. on day 0 and 28 with 2 doses ofPRPM (5 and 0.5 μg), non-adjuvanted or adjuvanted with 100 μg Adjuphos.The animals were challenged on day 49 with 10⁵ (pfu) of RSV A2. Animalswere sacrificed 5 days after challenge and titers were measured in lungsand nose.

Results

immunization with adjuvanted PRPM induced complete protection in thelungs and nose, with the exception of 1 animal that showed breakthroughin the nose. Most of the animals receiving 5 and 0.5 μg non-adjuvantedPRPM showed breakthrough in the lungs and noses and there was asignificant difference between the groups receiving the adjuvanted andthe non-adjuvanted protein (FIG. 8). The adjuvanted protein inducedsignificantly higher VNA titers compared to the non-adjuvanted proteinat day 49 after immunization (FIG. 9).

TABLE 1 Antibody sequences Ab VH domain VH CDR1 VH CDR2 VH CDR3 CR9501Amino acids 1-125 GASINSDNYYWT HISYTGNTYYTPSLKS CGAYVLISNCGWFDSof SEQ ID NO: 16 (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) CR9502Amino acids 1-121 GFTFSGHTIA WVSTNNGNTEYAQKIQ EWLVMGGFAFDHof SEQ ID NO: 18 (SEQ ID NO: 10) G (SEQ ID NO: 12) (SEQ ID NO: 11) AbVL domain VL CDR1 VL CDR2 VL CDR3 CR9501 Amino acids 1-107 QASQDISTYLNGASNLET QQYQYLPYT of SEQ ID NO: 17 (SEQ ID NO: 7) (SEQ ID NO: 8)(SEQ ID NO: 9) CR9502 Amino acids 1-110 GANNIGSQNVH DDRDRPS QVWDSSRDQAVIof SEQ ID NO: 19 (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15)

Sequences SEQ ID NO: 1: PRPMMELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAFRDGQXYYRKDGEWVLLSTFL SEQ ID NO: 2 PRQMMELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKTNQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 3 PRPM + S46GMELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL CR9501 heavy chain (SEQ ID NO: 16):QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNTYYTPSLKSRLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCCR9501 light chain (SEQ ID NO: 17):EIVMTQSPSSLSASIGDRVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSRFTGSGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCR9502 heavy chain (SEQ ID NO:18):EVQLLQSGAELKKPGASVKISCKTSGFTFSGHTIAWVRQAPGQGLEWMGWVSTNNGNTEYAQKIQGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCCR9502 light chain (SEQ ID NO: 19):QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSG1PDUSGSNSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTIAPTECSNucleotide sequence encoding PRPM (SEQ ID NO: 20):ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGGAAATCAAGTGCAACGGCACCGACGCCAAGGTCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGCGAGCTGCCCCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCTCTGCCATTGCTAGCGGCGTGGCCGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCTGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCTCCGTGTCATTCTTTCCACAGGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGTCCGCCATCGGCGGCTACATCCCCGAGGCCCCTAGAGATGGCCAGGCCTACGTGCGGAAGGACGGCGAGTGGGTGCTGCTGTCTACCTTCCTGNucleotide sequence encoding PRQM (SEQ ID NO: 21):ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGGAAATCAAGTGCAACGGCACCGACGCCAAGGTCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGCGAGCTGCCCCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCTCTGCCATTGCTAGCGGCGTGGCCGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCTGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCTCCGTGTCATTCTTTCCACAGGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGTCCGCCATCGGCGGCTACATCCCCGAGGCCCCTAGAGATGGCCAGGCCTACGTGCGGAAGGACGGCGAGTGGGTGCTGCTGTCTACCTTCCTGNucleotide sequence encoding PRPM + S46G (SEQ ID NO: 22):ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTTGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTATCTGGGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAAGAAATCAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGAAGAGAACTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTTCTGCTGGGAGTGGGAAGCGCCATTGCTAGCGGAGTGGCCGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCTCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAACAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCTGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGTCCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCTCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACAGAGGCTGGTACTGCGATAATGCCGGCTCCGTCTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAATCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACAAGCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGACGCCAGCATCAGCCAAGTGAACGAGAAGATCAACCAGAGCCTGGCMCATCAGAAAGTCCGATGAGCTGCTGAGCGCCATCGGCGGCTACATCCCTGAGGCCCCTAGAGATGGCCAGGCCTATGTGCGGAAGGACGGCGAATGGGTGCTGCTGTCTACCTTTCTG

The invention claimed is:
 1. A nucleic acid molecule encoding arecombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F)protein, the nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, andSEQ ID NO:
 22. 2. The nucleic acid molecule according to claim 1,wherein the nucleic acid molecule comprises the nucleotide sequence ofSEQ ID NO:
 20. 3. A vector comprising the nucleic acid moleculeaccording to claim
 2. 4. A composition comprising the nucleic acidmolecule according to claim
 2. 5. A method of inducing an immuneresponse against RSV F protein in a subject in need thereof, the methodcomprising administering to the subject the composition according toclaim
 4. 6. A method of prophylaxis and/or treatment of RSV infection ina subject in need thereof, the method comprising administering to thesubject the composition according to claim
 4. 7. A vaccine against RSVcomprising the nucleic acid molecule according to claim
 2. 8. Thenucleic acid molecule according to claim 1, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:
 21. 9. A vectorcomprising the nucleic acid molecule according to claim
 8. 10. Acomposition comprising the nucleic acid molecule according to claim 8.11. A method of inducing an immune response against RSV F protein in asubject in need thereof, the method comprising administering to thesubject the composition according to claim
 10. 12. A method ofprophylaxis and/or treatment of RSV infection in a subject in needthereof, the method comprising administering to the subject thecomposition according to claim
 10. 13. A vaccine against RSV comprisingthe nucleic acid molecule according to claim
 8. 14. The nucleic acidmolecule according to claim 1, wherein the nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO:
 22. 15. A vectorcomprising the nucleic acid molecule according to claim
 14. 16. Acomposition comprising the nucleic acid molecule according to claim 14.17. A method of inducing an immune response against RSV F protein in asubject in need thereof, the method comprising administering to thesubject the composition according to claim
 16. 18. A method ofprophylaxis and/or treatment of RSV infection in a subject in needthereof, the method comprising administering to the subject thecomposition according to claim
 16. 19. A vaccine against RSV comprisingthe nucleic acid molecule according to claim 14.